The mechanisms of thyroid hormone action at the cellular level will be explored with primary emphasis on the mitochondrial pathway. Our previous work has suggested that the active hormone triiodothyronine (T3) acts directly to stimulate mitochondrial oxidative phosphorylation. Out fractionation studies have yielded a protein arising from the inner mitochondrial membrane which appears to bind T3 specifically with an association constant (Ka) of approximately 2 x 1011 M-1, and which can be photoaffinity labeled with (125I)-T3. The T3 binding protein copurifies with adenine nucleotide translocase (AdNT) of inner mitochondrial membrane. Moreover the binding protein labeled with (125I)-T3 comigrates with AdNT on nondenaturing isoelectrofocusing gels and on 2D gels. Further evidence will be sought employing specific antisera against AdNT, peptide mapping, as well as studies of T3 binding specificity comparing congeners less or more active than T3. With these techniques we will compare the 31 kd protein most conspicuously photo-affinity labeled in intact mitochondria with purified AdNT, as well as characterizing the other proteins in mitochondria which we found showed some although less intense photoaffinity labeling. In functional studies we plan to study the effects of T3 on the nucleotide translocating activity of AdNT inserted into artificial liposomes to explore whether the effects involve interaction of T3 with AdNT alone or require other components of the mitochondrial membrane such as phospholipids. Preliminary work has suggested that T3 may cause markedly enhance phosphorylation of a 70 kd mitochondrial protein within 22 minutes, which raises the possibility that binding of T3 to mitochondria may initiate a complex cascade of events. Since we have observed prompt stimulation by T3 of ATP formation in isolated mitochondria, we plan metabolic studies of cultured T3 responsive cells to examine the early (derived from 2 hour) effects of hormone stimulation on oxygen consumption and ATP formation. Normal human liver cell lines (ARL 15 and Clone 9) on the activity and intracellular distribution of mitochondria will be examined with the vital mitochondrial fluorescent dye Rhodamine-123. The ultimate aim of the research is elucidation of the precise molecular mechanism whereby receptor occupancy leads to the physiological effects of the hormone.